Doses Of pFSH Research Articles

Comparison of three superovulation protocols with or without GnRH treatment at the time of artificial insemination on ovarian response and embryo quality in Thai native heifers.

To optimize the superovulation protocol in Thai native cattle, the present research was designed to (1) compare three different protocols designed to induce superstimulation and (2) study the effect of gonadotropin-releasing hormone (GnRH) administration at insemination time (to induce ovulation) on ovarian follicular activities in terms of the number of large follicles, corpora lutea (CLs) and unovulated follicles, and the number and quality of ova/embryos recovered in Thai native heifers. Initially, the estrous cycles of animals (n = 36) at unknown stages were synchronized by two prostaglandin F2α (PGF2α) injections at an interval of 12days. Follicular development of heifers was randomly superstimulated with one of three different treatment protocols: treatment A-a total of 100mg of pituitary-derived FSH (pFSH; Folltropin®-V) administered in eight decreasing doses; treatment B-a single dose of 100mg pFSH dissolved in 30% (w/v) polyvinylpyrrolidone; or treatment C-ablation of all follicles ≥5mm with a single dose of pFSH. All heifers received PGF2α 48h after the initiation of FSH treatment to induce luteolysis from the previous cycle, and they were twice inseminated at 12 and 24h after the onset of estrus. Heifers in each treatment were assigned to be injected or not with GnRH at the time of first insemination with frozen/thawed semen to induce ovulation. About 7days after artificial insemination (AI), ova/embryos were collected and classified. The numbers of large follicles at the onset of estrus were not statistically significantly different; meanwhile, the maximum diameters of follicles at the time of first insemination in treatment C were smaller compared with the other treatment groups (p < 0.001). The administration of GnRH at the first insemination time resulted in a greater number of CLs and fewer unovulated follicles at the time of ova/embryo collection (p = 0.001), which subsequently resulted in a higher number of total ova/embryos recovered (p = 0.030). Among heifers treated with different superstimulation protocols, the ablation of all follicles ≥5mm in diameter before superstimulation (treatment C) resulted in significantly higher quality of fertilized ova and transferable embryos (p = 0.001). In summary, it could be inferred that GnRH treatment improved ovarian function rather than embryo quality. Dominant follicle ablation prior to superstimulation is preferable for collecting a greater number of transferable embryos.

Efficiency analysis of standard and day 0 superovulatory protocols in Boer breed goats

ABSTRACT The aim of this research was to investigate the ovarian influence on the success of two superovulatory protocols in goats. Fifteen goats were used twice in the study in a crossover design: once within the control group where standard protocol was applied, and the other time in a modified, day 0 group, where no dominant follicle was present and the follicular cohort was homogenous (“Day 0 protocol”). All the animals were synchronized with vaginal pessaries and superovulated with 6 decreasing doses of pFSH. Although the day 0 protocol significantly decreased the presence of the dominant follicle before the start of the superovulation treatment; it did not improve the superovulatory success. Fifty-four percent of goats in the control group had a dominant follicle which exerted a negative influence on the number of transferable embryos. The other 46% of goats from the same group had a very good superovulatory response, which improved the overall response of the control group in comparison with the day 0 group. Another reason for the lower superovulatory success of the day 0 protocol was the smaller number of second and third category follicles (3.5 - 5.5 mm in size) at the onset of the superovulation protocol, that positively correlated with the number of the transferable embryos (r=0.67; P<0.05). In Boer goats, for the success of superovulatory treatment, the number of follicles of a certain size before the start of the superovulatory treatment has an effect as important as the effect of dominance. The application of the FSH hormone should start at the time when a suitable number of follicles belonging to the second category are present on the ovaries when performing the day 0 protocol. Key words: goat, superovulation, dominant follicle, follicular population, embryo, superovulatory success

Superovulation in wood bison (Bison bison athabascae): Effects of progesterone, treatment protocol and gonadotropin preparations for the induction of ovulation

Experiments were done to determine the ovarian response and embryo production following superstimulation of wood bison. In Experiment 1 (Anovulatory season), the efficacy of pLH vs. hCG for inducing ovulation was compared in wood bison superstimulated with a single dose of pFSH in 0.5% hyaluronan and the effect of exogenous progesterone (PRID) on superovulatory response and embryo quality was examined. In Experiment 2 (Ovulatory season), the efficacy of pLH vs. hCG for the induction of ovulation was compared in wood bison superstimulated with pFSH in a single intramuscular dose vs. a two-dose regimen 48h apart (split dose) in 0.5% hyaluronan. In Experiment 1, the number of CL was greater (P<0.05) in bison treated with hCG than pLH (6.6±1.8 vs. 2.8±0.8) and in those that were not given PRID (6.0±1.5 vs. 2.7±1.0). There was no effect of progesterone treatment on embryo quality. In Experiment 2, the number of CL was greater (P<0.05) in bison treated with hCG than with pLH (6.3±0.8 vs. 3.8±1.2) and in bison superstimulated with split dose vs. single dose of FSH (7.1±0.9 vs. 3.0±0.8). The number of ova/embryos and freezable embryos did not differ among groups in either experiment. In conclusion, hCG induced a greater ovulatory response than pLH in both seasons. Two doses of FSH induced the greatest superovulatory response during the ovulatory season. Exogenous progesterone did not improve embryo quality during the anovulatory season.

Effect of pFSH dose reduction on in vivo embryo production in Dorper ewes

&lt;p&gt;To evaluate the effect of pFSH dose on the &lt;em&gt;in vivo &lt;/em&gt;embryo production of Dorper ewes in the semi-arid northeast of Brazil, 40 sheep females were distributed into two groups of 20 animals that received intravaginal CIDR for 14 days, and two days before device removal, they received one of the following treatments: in the FSH200 group, the ewes received 200 mg of pFSH; and in the FSH128 group, the ewes received a total of 128 mg in decreasing doses every 12 h. Beginning 12 h after the conclusion of the treatments, estrus detection was performed every four hours using two Dorper rams of proven fertility. The ewes were mated at estrus onset and 24 hours later. Seven days after intravaginal device removal, the superovulatory response was evaluated, and embryo collection was performed using the laparotomy method. The recovered flushings were subjected to embryo searches under a stereomicroscope and classified according to their qualities. Analyses of variance (ANOVAs) and LSD tests were used to compare the different parameters. The data expressed as percentages were analysed by chi-square test. The ovulation rate was higher in the FSH200 group, which had 16.3 ± 0.3 corpora lutea (CL), than in the FSH128 group, which had 11.3 ± 0.3 CL (P&amp;lt;0.05). However, higher fertilization rate (83.6% vs. 62.4%) and higher transferable (86.0% vs. 71.6%) and freezable (67.9% vs. 40.8%) embryo rates were observed in the FSH 128 group compared with the group that received 200 mg. Furthermore, no significant differences in the remaining parameters were observed between the experimental groups (P&amp;gt;0.05), demonstrating that pFSH dose reduction promoted a greater production of freezable and transferable embryos in Dorper ewes subjected to MOET.&lt;/p&gt;

Effect of pFSH dose reduction on in vivo embryo production in Dorper ewes

To evaluate the effect of pFSH dose on the in vivo embryo production of Dorper ewes in the semi-arid northeast of Brazil, 40 sheep females were distributed into two groups of 20 animals that received intravaginal CIDR for 14 days, and two days before device removal, they received one of the following treatments: in the FSH200 group, the ewes received 200 mg of pFSH; and in the FSH128 group, the ewes received a total of 128 mg in decreasing doses every 12 h. Beginning 12 h after the conclusion of the treatments, estrus detection was performed every four hours using two Dorper rams of proven fertility. The ewes were mated at estrus onset and 24 hours later. Seven days after intravaginal device removal, the superovulatory response was evaluated, and embryo collection was performed using the laparotomy method. The recovered flushings were subjected to embryo searches under a stereomicroscope and classified according to their qualities. Analyses of variance (ANOVAs) and LSD tests were used to compare the different parameters. The data expressed as percentages were analysed by chi-square test. The ovulation rate was higher in the FSH200 group, which had 16.3 ± 0.3 corpora lutea (CL), than in the FSH128 group, which had 11.3 ± 0.3 CL (P&lt;0.05). However, higher fertilization rate (83.6% vs. 62.4%) and higher transferable (86.0% vs. 71.6%) and freezable (67.9% vs. 40.8%) embryo rates were observed in the FSH 128 group compared with the group that received 200 mg. Furthermore, no significant differences in the remaining parameters were observed between the experimental groups (P&gt;0.05), demonstrating that pFSH dose reduction promoted a greater production of freezable and transferable embryos in Dorper ewes subjected to MOET.

Effect of natural mating or laparoscopic artificial insemination in superovulated Santa Inês ewes on superovulatory response, fertility and embryo viability

This study evaluated the effect of two mating methods (GNM: natural mating or GAI: laparoscopic artificial insemination) on superovulatory response, fertility and embryo yield in superovulated ewes. Fifteen non-pregnant Santa Inês ewes were superovulated and either mated by GNM or GAI in a crossover design. Oestrus was synchronised using intravaginal progestagen sponges for 6 days and on Day 5, 300 IU eCG and 0.0375 mg d-cloprostenol were given. Twelve hours after sponge removal, 0.025 mg gonadotropin-releasing hormone was administered. Superovulation started 48 h after gonadotropin-releasing hormone treatment, using 5 IU/kg follicle-stimulating hormone (pFSH). At the first pFSH dose, new sponges were inserted. At the fifth dose, 0.0375 mg cloprostenol was administered and the sponges were removed. The GNM was mated with rams every 12 h, until the end of oestrus. The ewes of GAI were laparoscopic inseminated with frozen–thawed semen 36 and 48 h after sponge removal. Ultrasonography was performed every 24 h from the beginning of oestrus synchronisation treatment and every 12 h from the second sponge removal to 2 days after the last pFSH dose. Six to seven days after mating, the number of corpora lutea (CL) was evaluated by laparoscopy and the females with &gt; 4 CL were subjected to embryo collection. The interval from sponge removal to ovulation was shorter (P &lt; 0.05) in the GNM. The overall superovulatory response was 63.3% (19/30), with 60.0% and 66.7% in GNM and GAI, respectively (P &gt; 0.05). The number of recovered structures (6.4 ± 2.4 vs 4.5 ± 3.0), recovery rate (74.0 ± 16.0 vs 52.3 ± 26.5%), number of transferable embryos (3.0 ± 2.9 vs 3.6 ± 2.0) and viability rate (47.2 ± 45.3 vs 77.4 ± 37.1%) did not differ between GAI and GNM (P &gt; 0.05). However, the GAI group showed a higher (P &lt; 0.05) number of unfertilised oocytes (3.1 ± 3.1) and a higher non-fertilisation rate (47.1 ± 45.3%) than the GNM (0.9 ± 2.1 and 11.5 ± 21.5%). The mating method did not affect the superovulatory response, and production of viable embryos although the non-fertilisation rate has been inferior for the AI group.

Superovulatory response in Japanese Black cows receiving a single subcutaneous porcine follicle–stimulating hormone treatment or six intramuscular treatments over three days

To reduce labor for superovulation treatment by twice-daily intramuscular (im) administration of FSH for more than 3 to 4 days, we investigated the superovulatory responses of Japanese Black cows to porcine FSH (pFSH) used as a single subcutaneous (sc) administration at two different doses in two different volumes of saline. In experiment 1, 20 Armour units (AU) of pFSH dissolved in either 10 mL (treatment A; n = 14) or 50 mL (treatment B; n = 14) of saline was administered subcutaneously in the neck region. In experiment 2, 30 AU of pFSH dissolved in either 10 mL (treatment C; n = 15) or 50 mL (treatment D; n = 15) of saline was administered subcutaneously in the neck region. The control animals in experiment 1 (n = 14) and experiment 2 (n = 15) received 20 AU of pFSH administered intramuscularly twice daily in decreasing doses for more than 3 days. In experiment 1, mean (±SEM) numbers of CL (15.4 ± 2.5, 18.1 ± 3.4, and 17.2 ± 2.6), total number of ova and embryos (12.9 ± 1.4, 15.9 ± 3.5, and 16.2 ± 2.8), and transferable embryos (7.5 ± 2.0, 10.4 ± 2.8, and 8.0 ± 2.1) did not differ among treatments A, B, and control. In experiment 2, mean (±SEM) numbers of CL (20.5 ± 4.3, 20.4 ± 2.7, and 20.1 ± 3.4), total number of ova and embryos (21.7 ± 4.2, 17.3 ± 3.4, and 16.5 ± 3.2), and transferable embryos (8.1 ± 1.6, 9.3 ± 2.2, and 9.5 ± 1.9) did not differ among treatments C, D, and control. Although there were no differences in serum pFSH concentrations among the three treatments at each of the time points in experiment 1, in experiment 2, the serum pFSH concentration at 6 and 8 hours after pFSH administration in treatment C (3.1 ± 0.8, 2.7 ± 0.5 ng/mL, mean ± SEM) was significantly greater (P < 0.05) than in the control (0.7 ± 0.1, 1.1 ± 0.2 ng/mL). At 10 hours after administration, the pFSH concentration had decreased and there were no differences among the three treatments at subsequent time points. These results suggest that increasing the volume of saline or the dose of pFSH does not affect the absorption pattern of pFSH administered as a single sc administration. In conclusions, single sc administration of pFSH at a dose of 20 or 30 AU dissolved in 10 or 50 mL of saline is able to induce a superovulatory response comparable with that obtained by twice-daily im administration in Japanese Black cows.

Efficiency of superstimulatory protocol P-36 associated with the administration of eCG and LH in Nelore cows

Recent work with P-36 demonstrates that the replacement of the last two doses of Follicle-Stimulating Hormone (FSH) with equine chorionic gonadotropin (eCG) increases embryo yields. However, it is unclear if the positive effect of eCG is related to its FSH-like activity, LH-like activity, or both. This study aimed to verify the replacement of eCG with pLH on the last day of superstimulatory treatment. Twenty-five Nelore cows were allocated to four groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent four treatments in a crossover design. The control group cows were superstimulated with decreasing doses of porcine Follicle-Stimulating Hormone (pFSH, 133 mg, im). In the P-36/eCG, P-36/LH2, and P-36/LH4 groups, the last two doses of pFSH were replaced in the former group by two doses of eCG (200 IU each dose, im) and in the latter two groups by two doses of pLH (1 and 2 mg each dose, im), respectively. Donors received fixed-time artificial insemination 12 and 24 hours after pLH. Embryo flushing was performed on D16. Data were analyzed by ANOVA (Proc Mixed, SAS). There was a trend of decreasing ovulation rate when comparing groups LH2 and eCG (P = 0.06). However, there was no significant difference in the mean number of viable embryos among groups P-36 (3.3 ± 0.7), P-36/eCG (4.5 ± 0.5), P-36/LH2 (3.7 ± 0.8), and P-36/LH4 (4.2 ± 1.0). It is concluded that the replacement of eCG by pLH on the last day of superstimulatory treatment can be performed with no significant variation in the production of viable embryos.

Ovarian response to porcine FSH in association with ablation-induced or spontaneous follicular wave development during the estrous cycle in crossbred and Brazilian Warmblood mares

The primary objective of this study was to examine the follicular and ovulatory responses following treatment with pFSH in association with ablation-induced or spontaneous follicular wave emergence or follicle deviation during diestrus in crossbred (Mangalarga×Arabian) and Brazilian Warmblood mares with a propensity for spontaneous multiple ovulations; secondary considerations were given to the collection of embryos. In Experiment 1, crossbred mares were administered (im) saline (control, n=7) or pFSH (25mg) when the largest follicle of the ablation-induced follicular wave reached ≥13mm (n=7) or ≥20mm (n=7) or, after pre-treatment ovulation (Day 0) on Day 6 (n=7). In Experiment 2, crossbred mares were administered (im) saline (control, n=10) or a larger dose of pFSH (50mg, n=7) when the largest follicle of the ablation-induced follicular wave reached ≥13mm. In Experiment 3, Brazilian Warmblood mares were administered (im) saline (control, n=7), pFSH (25mg, n=7 or 50mg, n=5) or EPE (12.5mg, n=7) as a positive control on Day 6. Ultrasonic technology was used to ablate all follicles ≥8mm and to monitor follicular development and detect ovulation. Treatment with pFSH or EPE was done twice daily until the largest follicle reached ≥32mm; thereafter, hCG (2500IU) was administered (iv) when the largest follicle reached ≥35mm. Artificial insemination was done 12h after hCG and embryo collections were done 8 d after post-treatment ovulations. In Experiments 1 and 2, treatment of crossbred mares with pFSH post-ablation in association with the expected time of wave emergence or follicle deviation did not (P>0.05) enhance the follicular or ovulatory responses or collection of embryos compared to controls. In Experiment 3, although the enhanced ovulatory response of mares to EPE at the expected time of spontaneous wave emergence was not different (P>0.05) from controls, it was greater (P<0.05) than the response to pFSH. In conclusion, the novelty of using follicle ablation prior to pFSH treatment at the time of wave emergence or follicle deviation did not enhance the follicular or ovulatory responses or collection of embryos to treatment in crossbred mares. In addition, the hypothesis that Brazilian Warmblood mares with a greater propensity for spontaneous multiple ovulations are as responsive to pFSH compared to EPE was not supported. Thus, the combined experimental results of the present study continue to support the general consensus that pFSH is relatively ineffective for follicular superstimulation/superovulation in mares.

8 DURATION OF ESTRUS AND TIME OF OVULATION IN SUPEROVULATED SANTA INÊS EWES

The aim of this study was to determine the intervals between sponge removal and the onset of estrus, sponge removal and the first detected ovulation, and onset of estrus and the first ovulation, and also to determine the duration of estrus, in superovulated Santa Inês ewes subjected to natural mating (NM) and AI. The trial was done in July and February at Cachoeiras de Macacu–Rio de Janeiro (22°27′S, 43°39′W). Fifteen non-pregnant Santa Inês ewes, age 3.4 ± 1.4 years, weighing 47.8 ± 6.3 kg, with 3.3 ± 0.4 body condition score (scale of 1 to 5), were randomly assigned to 2 groups. Both groups were superovulated using the same protocol and were mated in a crossover design by NM and laparoscopic AI. Estrus was synchronized using intravaginal sponges (60 mg of medroxyprogesterone acetate; Progespon®, Schering Plough Animal Health, São Paulo, Brazil) inserted (Day 0) and maintained for 6 days. On Day 5, the ewes received IM injections of 300 IU of eCG (Novormon®, Schering Plough Animal Health) and 0.0375 mg of cloprostenol (Prolise®, Tecnopec, São Paulo, Brazil). Twelve hours after sponge removal, 0.025 mg of gonadorelin acetate (Gestran®, Tecnopec) was administered IM. Superovulation started 60 h after sponge removal and consisted of 5 IU kg–1 of porcine FSH (pFSH; Pluset®, Hertape Calier, Minas Gerais, Brazil) IM in 6 decreasing doses (25, 25, 15, 15, 10, and 10%) at 12-h intervals. At the first pFSH dose, new sponges were inserted. At the fifth pFSH dose, 0.0375 mg of cloprostenol was administered IM and the sponges were removed. After the sponge removal, the NM group was exposed to rams twice per day for mating, until the end of estrus. In the AI group, estrus was detected using a teaser with the penis diverted. The females were permitted to be mounted twice per day until the end of estrus, and were inseminated with frozen–thawed semen 24 and 36 h after the end of superovulation. The follicular development and ovulation time were observed using real-time ultrasonography (8.0 MHz Pie Medical®, Aquila Vet, Tokyo, Japan) at 12-h intervals. For statistical analysis, a Student’s t-test was performed (5% significance level) using the BioEstat program. Results are presented as mean ± standard deviation. The time from sponge removal to onset of estrus and the duration of estrus did not differ between NM and AI groups (31.79 ± 5.94 v. 25.25 ± 10.38 h and 29.89 ± 11.54 v. 26.66 ± 8.67 h, respectively). The time from sponge removal to ovulation and from onset of estrus to the first ovulation were shorter (P &lt; 0.001) in the NM group (32.11 ± 12.72 v. 56.48 ± 15.39 h and 8.61 ± 5.99 v. 32.25 ± 18.57 h, respectively). The time from sponge removal to ovulation in July and February was 56.07 ± 7.27 versus 56.83 ± 20.72 in the AI group and 29.54 ± 0.56 versus 33.83 ± 19.02 in the NM group, respectively, suggesting that the season of the year in a tropical region did not influence the ovulation time for each treatment. Possibly, the mechanical stimulation induced by the contact of the penis with the vagina fornix and by the accessory sex glands fluids in mating hastened the ovulation time in the NM group. The service can shorten the time of ovulation.

Maximising embryo production in endangered sheep breeds: in vitro procedures that complement in vivo techniques

This study investigated the use of previously superovulated ovaries as a source of oocytes, assessing the competence of them for in vitro embryo production. Two superovulatory treatments were performed: equine Chorionic Gonadotrophin (eCG) plus porcine Follicle-Stimulating Hormone (pFSH) in a single dose or the conventional protocol of six decreasing doses of pFSH. Thirty donor ewes of the endangered Ojalada breed were given either the simplified (group S; n=15) or the decreasing-dose (group D; n=15) treatments three times at intervals of ≥50 days. Ovaries were recovered on day 7 after the oestrus following the third treatment, just after embryo flushing, and the oocytes were collected to assess in vitro maturation, fertilisation and development to the blastocyst stage. The two superovulatory treatments did not differ in the mean number of oocytes selected for maturation (7.1±1.2 and 8.5±1.5 per ewe in the D and S groups, respectively). The oocytes recovered from ewes in Group D (87.5%) had a significantly (p<0.05) higher maturation rate than did those recovered from ewes in group S (75%), but no differences were found in fertilisation rate (94% and 94.6% in the D and S groups, respectively); both groups did not differ in their blastocyst rates and the total number of cells in in vitro-produced blastocysts. In the two experimental groups, 1.7 (D) and 1.8 (S) in vitro-produced blastocysts were generated per ewe, which indicate that it is feasible to combine in vivo and in vitro techniques to maximise embryo production in endangered sheep breeds.

The effect of exogenous gonadotropins on ovarian function in goats actively immunized against inhibin

The aim of this investigation was to compare the ovarian response to superovulatory treatments in does before and after inhibin immunization, with a view to optimizing the superovulatory potential of the caprine ovary. To avoid interference by the ovarian cycle, the experiment was conducted out-of-season. At the onset of the experiment 48 does were subjected to treatment with an sc implant of the progestogen norgestomet, combined with a gonadotropin; eight does each received a single injection of 1200 IU eCG, 400 IU eCG or 2 mL physiological saline (control) or six injections (at 12 h intervals) constituting 16 or 5.4 AU pFSH. The does were mated and subjected to embryo collection 6 to 7 d later. Throughout the experiment ovarian function (by ultrasonography) and plasma levels of inhibin antibodies and progesterone were monitored. Of 40 does treated during the first part of the experiment, 48% showed estrus. The ovarian response in does treated with a high or low dose of eCG or a low dose of pFSH was barely in excess of the ovarian response in the saline-treated controls, whereas a superovulatory dose of pFSH (16 AU) gave a satisfactory response of, on average, 14.5 ovulations (yielding 8.8 flushed ova and embryos). Immediately after the does had been subjected to embryo collection they were actively immunized against inhibin by administering two injections of a recombinant α-subunit of ovine inhibin at four week intervals. All immunized does produced antibodies with the maximal titer reached two weeks after the second injection. Groups of immunized does were subjected to the same gonadotropin treatments as before (avoiding allocation of individuals to the same treatments). This time all does showed estrous symptoms. The ovulatory response to the various treatments, including the saline controls, was virtually identical, the overall average being 21.8 follicles and 9.1 ovulations. The average embryo yield per doe was 5.7. The results imply that inhibin acted as the key factor in determining the ovulatory response since no impact of any of the supplementary gonadotropins was noted in inhibin-immunized does. This finding gives rise to the notion that inhibin antibodies may act primarily by an intraovarian paracrine action rather than by reducing the suppressive action of inhibin on pituitary FSH release. Further, these findings confirm earlier reports that eCG is less suitable than FSH for inducing superovulation in goats, and indicate that active immunization against inhibin may be considered a viable alternative to using exogenous gonadotropin for inducing superovulation in goats.

Improvement of embryo production by the replacement of the last two doses of porcine follicle-stimulating hormone with equine chorionic gonadotropin in Sindhi donors

The aim of this study was to evaluate the superovulatory (SOV) response of Sindhi ( Bos indicus) donors submitted to an ovarian follicular superstimulatory protocol replacing the last two doses of pFSH by eCG. Forty-eight SOV treatments were performed in a crossover design in 19 nulliparous and primiparous females that were randomly divided into two groups: FSH ( n = 24), which consisted of eight pFSH injections, or FSH/eCG ( n = 24), which consisted of six pFSH injections followed by two eCG injections. Each female underwent two or three SOV treatments that consisted of an i.m. injection of 2 mg estradiol benzoate and the insertion of an intravaginal progesterone-releasing device on Day 0. On Day 4, superstimulatory treatments were initiated and 100 mg pFSH was divided into twice daily decreasing doses over a 4-day period. In the FSH/eCG group, the last two doses of pFSH were replaced by two doses of eCG (150 IU eCG each). At the time of the fifth and sixth injections of FSH, 0.150 mg PGF 2α was injected i.m. The intravaginal progesterone-releasing device was removed at the time of the last FSH or eCG injection and ovulation was induced with 0.2 mg GnRH 18 h later. All females were artificially inseminated with frozen–thawed semen from the same bull 6 and 18 h after GnRH treatment. Seven days after GnRH treatment, embryos/ova were recovered and classified. Follicular superstimulatory (number of follicles ≥6 mm at the time of the last FSH or eCG injection) and SOV (CL number) responses were determined by transrectal ultrasonography. Data were analyzed using generalized linear models and results were presented as least squares means ± standard error. The FSH/eCG group had higher superstimulatory (33.8 ± 3.9 compared to 23.8 ± 2.6 follicles; P = 0.03) and SOV (16.8 ± 2.9 compared to 10.8 ± 2.1 CL; P = 0.10) responses. Although the number of total ova/embryos was not different between groups (8.2 ± 1.8 compared to 5.9 ± 1.4 for FSH/eCG and FSH groups, respectively; P = 0.25), the number (5.8 ± 1.3 compared to 2.6 ± 0.7; P = 0.02) and percentage (75.6 ± 5.7 compared to 53.2 ± 9.7%; P = 0.05) of transferable embryos was greater for the FSH/eCG females. Therefore, there was improvement in follicular superstimulatory and SOV responses and embryo quality in FSH/eCG-treated females.

318 EFFICIENCY OF PROTOCOL P-36, ASSOCIATED WITH EQUINE CHORIONIC GONADOTROPIN OR LUTEINIZING HORMONE ADMINISTRATION, IN THE LAST DAY OF SUPERESTIMULATORY TREATMENT, IN NELLORE COWS

The objective of this study was to evaluate the use of pLH in replacement of eCG on the last day of P-36 superstimulatory treatment in Nellore donors. Recent studies demonstrated improvement in embryo production when the last 2 doses of FSH were replaced by eCG. However, consecutive use of eCG in bovine superstimulatory protocols may induce antibody against eCG, decreasing embryo production. Twenty-five Nellore cows were randomly allocated in 4 groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent 4 treatments in a cross-over design. Donors received an intravaginal device containing 1.0 g of progesterone (IVD, Primer®, Tecnopec, Sao Paulo, Brazil) and oestradiol benzoate (2.0 mg, IM; Estrogin®, Farmavet, Sao Paulo, Brazil) at a random stage of the oestrous cycle (D0). Cows from the control group were superestimulated with decreasing doses of pFSH (133 mg, IM; Folltropin-V®, Bioniche, Ontario, Canada; D5-8). In the P-36/eCG group, the last 2 doses of pFSH were replaced by 2 doses of eCG (200 IU each dose, IM; Folligon®, Intervet, Boxmeer, the Netherlands). In the P-36/LH2 and P-36/LH4 groups, the last 2 doses of pFSH were replaced by 2 doses of 1 and 2 mg of pLH, respectively (IM; Lutropin®, Bioniche). All animals were treated with prostaglandin F2α (150 μg d-cloprostenol, IM; Prolise®, Tecnopec) on D7, and the IVD was removed 36 h after. Ovulation was induced with 12.5 mg of pLH (IM) on D9, and all animals received fixed-time artificial insemination 12 and 24 h after pLH. Embryo flushing was performed on D16. Data were analysed by ANOVA (Proc Mixed, SAS). There was a significant difference in the number of corpus luteum in the eCG group (19.2 ± 2.4) when compared with the LH2 (12.7 ± 2.0) and LH4 groups (12.3 ± 1.5; P &lt; 0.05). In addition, there was a tendency of lower ovulation rate in the LH2 group as compared with the eCG group (50.6 and 67.8%, respectively; P = 0.06). However, there was no difference in viable embryo yield among groups P-36 (3.3 ± 0.7), P-36/eCG (4.5 ± 0.5), P-36/LH2 (3.7 ± 0.8), and P-36/LH4 (4.2 ± 1.0); P &gt; 0.05. In conclusion, eCG can be replaced by pLH (4.0 mg), in the last day of P-36 protocol, without affecting the production of viable embryos in Nellore cows. The authors acknowledge FAPESP (Sao Paulo, Brazil) for funding and fellowships for A. C. S. Oliveira, M. C. C. Mattos, and M. R. Bastos.

Attempts at Superovulation of Mares With Porcine Follicle Stimulating Hormone and Recombinant Equine Follicle Stimulating Hormone

Attempts to superovulate mares have been disappointing and expensive. Conflicting data exist on the effectiveness of porcine follicle stimulating hormone (pFSH) as a superovulatory treatment for horses. Recently, a recombinant equine FSH (reFSH) has become available with covalently linked alpha and beta subunits, which results in a longer half-life than endogenous FSH. The purpose of this study was to compare doses of pFSH and reFSH for superovulating mares. Twenty-nine mares received injections of 25, 50, 100, or 150 mg pFSH or 0.5 mg reFSH 2 times per day. Mares were used up to three times, with their second reproductive cycle serving as an untreated control. All treated mares were administered cloprostenol on the second day of treatment and given 2,500 IU of human chorionic gonadotropin 24 to 38 hours after the majority of large follicles were >30 mm. Mares with untreated control cycles also received cloprostenol, but deslorelin was used to induce ovulation. No response from superstimulation treatments differed ( P > .1) from those of controls; mean ovulations per cycle ranged from 0.85 to 1.31; mean embryo recovery rates ranged from 0.66 to 1.08. Two of the eight mares treated with reFSH failed to ovulate. Porcine FSH was ineffective at inducing multiple ovulations at any dose. Although previous studies of reFSH yielded high ovulation rates, further research is needed to establish optimal protocols and to determine the cause of failed ovulations.

Superovulatory Response of Zebu Cows Treated with pFSH in a Single Subcutaneous Injection Followed by an Additional Intramuscular Sub‐Dose 48 h Later

The aim of this study was to evaluate whether an additional intramuscular (im) injection of pFSH would increase the embryo production in zebu cows superovulated with a single subcutaneous (sc) pFSH injection. Twenty-one Nelore cows were treated with a progesterone vaginal implant (Controlled Internal Drug Relased - CIDR B) and injected im with 2.5 mg estradiol benzoate. Four days later, cows were assigned randomly into three groups and superovulated with pFSH. Groups A and B received single sc injections of 400 and 320 IU, respectively; Group C received multiple im injections of 400 IU in decreasing doses at 12-h intervals over 4 days. In the morning (07:00 am) of day 3 after starting superovulation, cows received im 150 mcg cloprostenol and Group B was additionally injected im with 80 IU of pFSH. CIDR-B was withdrawn in the afternoon (07:00 pm). Cows were inseminated 48 and 62 h after the cloprostenol injection. Embryo collection and corpora lutea (CL) estimation were done 7 days after insemination. Alternation of treatments (crossover design) occurred at a 60-day interval. There was no significant difference (p > 0.05) of CL counts among treatments. The total (transferable and no transferable) number of recovered embryos from Group A (6.9 +/- 1.5) was not different from Group C (9.8 +/- 1.2), whereas Group B (5.7 +/- 1.5) was lower than Group C (p < 0.05). The number of transferable embryos from Groups A (2.4 +/- 0.7) and B (1.7 +/- 0.6) was lower (p < 0.05) than Group C (4.6 +/- 1.2). Lesser (p < 0.05) embryo production from Group B was related to lower recovery rate (46.4%), compared with Groups A (65.1%) and C (81.7%). It was concluded that an additional im subdose of pFSH, injected 48 h after a single subcutaneous (sc) dose of pFSH, does not improve the embryo production in zebu cows.

414 IMPROVEMENT OF EMBRYO QUALITY BY THE REPLACEMENT OF THE LAST TWO DOSES OF PORCINE FOLLICLE-STIMULATING HORMONE BY EQUINE CHORIONIC GONADOTROPIN IN SUPERSTIMULATED SINDI DONORS

Results from a previous experiment (Barros C et al. 2008 Reprod. Fertil. Dev. 20, 152 abst) have shown that the replacement of porcine FSH (pFHS) by eCG on the last day of the superstimulatory treatment in Nellore (Bos indicus) cows resulted in a greater superovulatory (SOV) response as compared with treatment exclusively with pFSH. However, another study (Sartori R et al. 2009 Reprod. Fertil. Dev. 21, 245-246 abst) in Nellore heifers did not corroborate those observations. The aim of this study was to evaluate the SOV response of Sindi (Bos indicus) donors submitted to this protocol. Forty-eight SOV treatments were performed in a crossover design in 19 nulliparous and primiparous females that were randomly divided into 2 groups: FSH (n = 24), which consisted of 8 pFSH injections (Folltropin®-V, Bioniche Animal Health; Belleville, Ontario, Canada), or FSH-eCG (n = 24), which consisted of 6 pFSH injections followed by 2 eCG injections (Folligon®, Intervet Schering-Plough Animal Health, Brazil). Each female underwent 2 or 3 SOV treatments that consisted of an i.m. injection of 2 mg of estradiol benzoate (Gonadiol®, Intervet) and the insertion of an intravaginal progesterone-releasing device (DIB®, Intervet) on Day 0. On Day 4, the superstimulatory treatments (100 or 150 mg of pFSH, based on previous history of SOV responses) were initiated and given in decreasing doses twice a day over a 4-day period. In the FSH-eCG group, the last 2 doses of pFSH were replaced by 2 doses of eCG (150 or 200 IU of eCG each). At the time of the fifth and sixth injections of FSH, 0.150 mg of PGF2 (Preloban®, Intervet) was injected i.m. The DIB® device was removed at the time of the last FSH or eCG injection and ovulation was induced with 0.2 mg of GnRH (Fertagyl®, Intervet) 12 h later. All females were artificially inseminated with frozen-thawed semen from the same bull 12 and 24 h after GnRH treatment. Seven days after the first AI, embryos/ova were recovered. Superstimulatory (number of follicles ≥6 mm at the time of the last FSH or eCG injection) and SOV (CL number) responses were determined by transrectal ultrasonography. Data were analyzed using generalized linear models and results were presented as least squares means ± standard error. The FSH-eCG group had higher superstimulatory (33.3 ± 3.9 v. 24.2 ± 3.0 follicles; P ≤0.06) and SOV (16.8 ± 2.9 v. 10.8 ± 2.1 CL; P ≤ 0.10) responses. Although the number of total ova/embryos recovered was not different between groups (8.2 ± 1.8 v. 5.9 ± 1.4 for FSH-eCG and FSH groups, respectively; P &gt; 0.25), the number (6.5 ± 1.2 v. 2.4 ± 0.7; P ≤ 0.008) and percentage (75.2 ± 6.6 v. 52.8 ± 8.6%; P ≤ 0.05) of viable embryos was greater for the FSH-eCG females. Contrasting with the results in Nellore heifers, this study observed improvement in superstimulatory and SOV responses and embryo quality by replacing the last 2 doses of pFSH by eCG in Sindi donors. Financial support from FAPESP, EMBRAPA, Intervet Schering-Plough Animal Health, Nutricell, and Sindi do ACS of Brazil.

167 EVALUATION OF SUPEROVULATORY RESPONSE IN Santa Inês EWES BY ULTRASONOGRAPHY AND LAPAROSCOPY

The development and use of non-invasive techniques would reduce risks of surgery sequels on the same animal and use of the donor could be optimized. The aim of this study was to evaluate the accuracy of ultrasonography performed prior to embryo collection to estimate superovulation response in sheep. Fifteen pluriparous Santa Inês sheep, 2 to 5 year old, with an average body weight of 46.79 ± 6.00 kg and body condition score of 2.96 ± 0.32 (1 to 5 scale) were allocated into 3 groups (GI, GII, GIII) in a cross-over design. In GI, intravaginal sponges (60 mg of medroxyprogesterone acetate; Progespon®, Schering Plough Animal Health, São Paulo, Brazil) were inserted (Day 0) and maintained for 14 days, and the superovulatory (SOV) protocol started on Day 12. In GII and GIII, intravaginal sponges were inserted (Day 0) and maintained for 6 days. On Day 5, the animals were treated with 300 IU of eCG (Novormon 5000®, Schering Plough Animal Health) and 5 mg of dinoprost (Lutalyse® Pfizer Animal Health, São Paulo, Brazil) i.m. Animals in GIII received 0.025 mg of gonadorelin acetate (Gestran-Plus®, Tecnopec, São Paulo, Brazil) i.m. 12 h after sponge withdrawal. In GII and GIII, the SOV protocol started 48 h after sponge removal and a new sponge was inserted immediately after its removal. The SOV protocol in all groups consisted of 200 mg of pFSH (Folltropin-V®, Tecnopec) administered in 6 decreasing doses given every 12 h (50/50, 30/30, and 20/20 mg). At the time of the fifth dose of pFSH, 5 mg of dinoprost was administered i.m. and the sponges were removed. Animals were bred by a sexually mature ram twice a day until the end of estrus. Prior to embryo collection, an ultrasonographic evaluation (5 MHz, Aloka SSD-500, Tokyo, Japan) was performed to determine the number of CL present on both ovaries. The number of CL was further evaluated by laparoscopy, when it was possible to detect poor responders or even non-ovulating animals. Statistical analysis was performed using all tests at the 95% confidence interval by SAEG program. Results are presented as mean ± SE. The number of CL was not different among all groups. The total number (GI, GII, and GIII) of CL determined by ultrasonography (9.09 ± 5.01) was not different (P &gt; 0.05) from that observed by laparoscopy (8.87 ± 5.25). A significant correlation (r = 0.56, P &lt; 0.0005) between evaluation performed by ultrasonography and laparoscopy was observed. These results suggest that ultrasonography can be used to determine the response to the superovulatory protocol. As embryo collection in sheep is performed mainly by surgical techniques, the implementation of non-invasive techniques such as ultrasonography could avoid unnecessary surgeries on animals that did not respond to the SOV protocol, therefore preventing early culling of embryo donors. Financial support: Fopesq/Proppi and Faperj (E26/171.065/2006).

200 EVALUATION OF SUPEROVULATORY REGIMES FOR IN VIVO EMBRYO PRODUCTION IN ALPACAS (LAMA PACOS)

The objective of the study was to evaluate 4 superovulatory regimes in terms of the quantity of transferable embryos recovered. A total of 48 female alpacas, 3 to 5 years of age and located at Malkini Alpacas Farm (4100 m elevation), were distributed into 4 treatments. In treatment 1, 13 female alpacas received on Day 0 an intravaginal device containing 0.78 mg of progesterone (Cue Mate®, Bioniche Animal Health, Belleville, Ontario, Canada) followed immediately by an i.m injection of estradiol (1 mg of estradiol benzoate) and an i.m. injection of PGF2α (Veyx®, 0.25 mg of cloprostenol). The intravaginal device was removed on Day 7, performing at removal time an i.m. injection of estradiol. From Days 8 to 16, the alpacas received an i.m injection twice per day and 12 hours apart of pFSH (FolltropinV®, Bioniche Animal Health) in decreasing doses totaling 420 mg of pFSH; on Day 16,300 IU of eCGi.m. (Pregnecol®, Bioniche Animal Health) was injected. In treatment 2, 13 alpacas received on Day 0 an intravaginal device of progesterone followed by an i.m. injection of PGF2; from Days 5 to 9, alpacas received injections twice per day of decreasing doses of pFSH (porcine FHS) totaling 320 mg; on Day 7, the intravaginal device was removed and 500 IU i.m. of eCG was injected. In treatment 3,13 alpacas received on Day 0 an intravaginal device of progesterone followed immediately by an i.m injection of GnRH (Conceptal®, 0.0042 mg of acetate of busereline); pFSH was injected i.m. from Days 5 to 9 in decreasing doses twice per day, totaling 440 mg; the intravaginal device was removed on Day 7. In treatment 4, 9 female alpacas received on Day 0 an i.m. injection of GnRH after verifying the presence of a preovulatory follicle (&gt;8.0 mm diameter). On Day 2, the alpacas received 1000 IU i.m. of eCG followed on Day 7 by an i.m. injection of PGF2. In all cases, the donor alpacas were evaluated by ultrasonography. The matings for treatments 1, 2, and 3 were performed twice per donor alpaca at 12-hour intervals between Days 5 and 8 of the initiation of the pFSH treatments, whereas in treatment 4 the matings were made the following day after the application of the PGF2. In treatment 1, the donor alpacas received at time of first mating an i.m injection of 3.75 mg of LH (Lutropin®, Bioniche Animal Health); in treatments 2, 3, and 4, the donors received an i.m. injection of GnRH. In all treatments, embryo collection was performed by nonsurgical method 6.5 days after first mating. There were significant differences between treatments (P &lt; 0.05) in the mean number of CL, with treatment 4 being the highest (4.7 ± 2.63, 4.1 ± 3.05, 1.8 ± 1.8, and 6.0 ± 3.16 for treatments 1 to 4, respectively). The total number of blastocysts recovered per treatment was 7, 16, 2, and 18 for treatments 1 to 4, respectively. The superovulatory strategy followed for treatment 4 showed to be the one resulting in the highest number of transferable embryos. Further comparative evaluations between FSH and eCG treatments are recommended. Research was partially funded by the contributions of Bioniche Animal Health.

Relationship between circulating concentrations of ovarian steroids and the superovulatory responses in anestrous ewes following a multiple-dose pFSH regimen

It has been suggested that elevated concentrations of ovarian steroids during superovulatory treatment may be a cause of poor superovulatory yields in ewes. The aim of this study was to examine the correlations between daily serum concentrations of estradiol-17β (E 2-17β) and progesterone concentrations, ovarian responses and in vivo embryo production in anestrous ewes superovulated in a multiple-dose pFSH regimen. Twelve clinically healthy Rideau Arcott ewes were used in the study (May–June). Medroxyprogesterone acetate (MAP)-releasing intravaginal sponges (60 mg) were inserted for 14 days, and 12 days after insertion of the sponges, 6 im injections of pFSH (Folltropin ®-V) were administered (1 × 2.5 ml and 5 × 1.25 ml) at 12-h intervals. Injections were administered at 20:00 and 08:00 h, beginning on Day 12 and finishing on Day 15 following sponge insertion. At the first pFSH treatment, all ewes received a single im injection of eCG (500 IU). Sponges were removed at the time of the 5th pFSH injection, and on the evening of Day 15 all ewes received an im injection of GnRH (50 μg). Ewes were then placed in an enclosed paddock with 2 adult Rideau Arcott rams for a 36-h period and embryos were recovered surgically 7 days after the GnRH injection. Daily blood samples (14:00 h) were drawn for the 10-day period from the onset of ovarian superstimulation. Serum concentrations of E 2-17β on the day of the 1st pFSH dose were negatively correlated to the numbers of embryo (Grades 1–3) ( r = −0.62; P < 0.05) and embryo viability rate ( r = −0.84, P < 0.01). The total number of luteal structures was positively correlated to serum E 2-17β concentrations on the 2nd and 3rd day after the onset of the superovulatory treatment (both r = 0.59, P < 0.05). There were no significant correlations between serum progesterone concentrations, from 1 to 7 days after the GnRH injection, and superovulatory responses. These findings suggest that serum E 2-17β concentrations may be more critical during follicular development than early embryonic development for determining embryo yield and viability, and may be responsible for some of the variation experienced in superovulatory regimens in ewes.